A major component of barrier function in stratified squamous epithelia is the cornified cell envelope (CE). This is a multi-component 10 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the cross-linking of several structural by transglutaminases. Studies on the biology and assembly of the protein and lipid components are a major effort of this laboratory. Specifically, we are studying: (i) the cross-linking of proteins in CEs isolated from a variety of sources to explore which proteins are cross-linked together through which glutamines and lysines, and to provide information on structure and function; (ii) several key structural proteins such as loricrin, the small proline rich protein (SPR) families, involucrin, envoplakin and periplakin; (iii) the ceramide lipids which become covalently attached to the CE; (iv) the earliest stages of CE assembly produced in cultured keratinocytes; and (v) an attempt to recreate a CE-like structure using an in vitro synthetic lipid vesicle (svi) model system. CE protein envelope structure and assembly. (1) During this year our long-time efforts toward obtaining structural information on one of the components of cornified cell envelope barrier structure in stratified squamous epithelia, periplakin, met with success. Rotary-shadowed images of purified protein revealed very flexible 85-90 nm-long rod-like molecules. Antibodies against C-terminus of periplakin labeled only one end of the molecule, providing evidence for parallel alignment of two chains in predicted double stranded coil-coil for the first time. (2) Cellular interactions of periplakin have been characterized by immunofluorescent analysis in cultured human keratinocytes and by in vitro experiments. Its N-terminal domain turned out to be responsible for binding to filamentous actin, while C-terminal domain showed selective binding to keratins 8 and 14, components of intermediate filaments network in simple and stratified epithelia correspondingly. Elongated shape of periplakin molecule revealed by electron microscopy should allow it to efficiently interconnect actin microfilament and the intermediate filament networks, playing role in cell network integration. (3) Experiments done in collaboration with an investigator at Jefferson Medical College, showed that caspase 6, which is activated during apoptosis, specifically cleaves periplakin close to C-terminus, effectively separating its intermediate filaments binding part from domain which is responsible for actin binding (A. E. Kalinin). It remains to be investigated whether this event plays a role in terminal differentiation of cells in stratified epithelia. (4) To further characterize alignment of individual chains in periplakin oligomeric molecule, series of cross-linked peptides from the oligomer were generated and analyzed by amino acid sequencing. (5) To obtain 3D structural information on periplakin domain responsible for intermediate filaments binding, series of GST-fusion constructs of periplakin, containing this C-terminal region, have been expressed in bacteria and purified. Crystallization trials are ongoing.